出芽短梗霉F4的溶磷能力及机理

从安徽省铜陵市铜官山尾矿库木贼根际分离筛选出的出芽短梗霉F4,以磷酸钙、磷酸铝、磷酸铁和磷矿粉4种不同磷源进行液体培养,测定培养液的pH、水溶性磷、菌体磷及有机酸含量.结果表明： 菌株F4对不同磷源的溶磷能力为：磷酸铝>磷酸铁、磷酸钙>磷矿粉,溶磷量均高于200 mg·L^-1;培养液pH在48 h内迅速下降,以磷酸铝、磷酸铁为磷源的培养液pH下降幅度明显大于磷酸钙与磷矿粉.出芽短梗霉F4产生的有机酸主要为草酸、柠檬酸和酒石酸,其中,以草酸为主.菌株的溶磷能力与有机酸无显著相关性,而与pH呈显著相关.接种出芽短梗霉F4时加入葡萄糖,尾矿中速效磷含量显著增加,说明出芽短梗霉F4在尾矿生态修复中具有潜在的应用价值.
     

Ascorbic acid specific utilization by some yeasts

One hundred and eighty strains of yeasts belonging to 17 genus and 53 species were screened for their ability to grow on ascorbic acid and iso-ascorbic acid as the sole carbon source. Most of the tested strains (157) were unable to grow on either compound. Strains of seven species of the genus Cryptococcus, of two Candida species, of Filobasidiella neoformans, Trichosporon cutaneum, Lipomyces starkeyi, Hansenula capsulata, and one strain of Aureobasidium pullulans were able to grow on ascorbic as well as on iso-ascorbic acid. Conversely, four strains of Aureobasidium pullulans, Candida blankii, and Cryptococcus dimennae could use only ascorbic acid for growth.     

表面活性剂对出芽短梗霉多糖生产影响的研究

研究了表面活性剂对出芽短梗霉细胞培养过程中多糖释放的影响。在摇瓶中,比较添加0.05%(w/v)的Tween 80、Tween 60、Tween 40,结果显示几种表面活性剂均能促进细胞释放多糖,其中以Tween 80的效果最佳。在5L发酵罐中,以100g/L玉米粉水解液做碳源的出芽短梗霉细胞培养液中分别添加了表面活性剂Tween 80 0.01%、0.05%、0.1%,其中以添加Tween 800.05%时的效果最好,与不添加表面活性剂相比多糖产量提高25%左右,发酵周期缩短了将近2d。     

Polysaccharide production by sponge-immobilized cells of the fungus Aureobasidium pullulans

T.P. WEST AND B.R.-H. STROHFUS. 1996. Cells of the fungus Aureobasidium pullulans ATCC 42023 were immobilized in sponge cubes and examined for their ability to elaborate the polysaccharide pullulan in relation to carbon source. It was found that fungal cells grown on corn syrup, sucrose or glucose as a carbon source could be immobilized in sponge cubes and that comparable cell weights and viable cell concentrations were immobilized. Independent of the carbon source tested, the immobilized fungal cells could be used at least three times for the production of polysaccharide. The immobilized A. pullulans cells elaborated the highest polysaccharide levels in the culture medium after 5–7 d of growth at 30°C.     

Effect of environmental factors and precursors on oxalic acid production,mycelial biomass and virulence of a potential bioherbicide isolate of Sclerotium rolfsii SC64 produced in liquid culture

The fungus Sclerotium rolfsii is presently under development as a bioherbicide for broadleaf weed species using fungus-infested substrates as application material in this laboratory. The effect of environmental factors and three precursors (citric acid, ascorbic acid, and sodium succinate) on mycelial growth, oxalic acid production, and virulence by SC64 in liquid culture were investigated. The results showed that for mycelia growth the optimum liquid medium was Modified Richard's solution (MRS) among the five tested media, but potato dextrose broth (PDB) produced the maximum oxalic acid production and virulence on detached Solidago canadensis leaves. When PDB was used as the basic medium, the oxalic acid/mycelial dry weight (mg g^–1) ratio reached the peak 4 days after inoculation. The optimum temperature for oxalic acid production was at 27°C, but increased mycelial dry weight and virulence were observed at 30°C. The optimum range of initial pH value for oxalic acid accumulation was 4.0–6.0, with the optimal pH 5.0; highest mycelial growth was with an initial pH 3.5–6.0 (optimum pH 5.0) and subsequently pH 3.5–5.5 (maximum at pH 3.5). Both mycelial dry weight and oxalic acid production showed a decreasing trend as a result of the precursor of oxalic acid being added to PDB. Among the three precursors, the greatest decrease in mycelial dry weight, and oxalic acid production was caused by sodium succinate. This clarification of optimal conditions for production of mycelial biomass while insuring high concentrations of oxalic acid and high virulence should be useful for further development of this fungus as biocontrol agent.     

控制pH环境对出芽短梗霉胞外多糖合成的影响

采用添加 Ca CO₃ 和 HCl的方法研究了 p H对出芽短梗霉多糖发酵的影响规律。在 P2培养基中发酵2 4 h,该菌有一个强烈的产酸期 ,导致 p H迅速下降到 3.6左右。在此 p H环境下继续发酵 12 0 h,多糖产量仅为 5.9g/ L。如果用 MP2培养基 (P²⁺ 0 .5% Ca CO₃ )发酵 ,由于 Ca CO₃ 缓冲了发酵 p H的下降 ,在整个发酵过程中 p H值可以维持在 5.0以上 ,多     

出芽短梗霉产聚苹果酸发酵条件的优化

【背景】出芽短梗霉可发酵葡萄糖生成聚苹果酸,但存在转化率和转化效率低等瓶颈,阻碍其实现商业化生产。【目的】通过优化发酵培养条件,提高出芽短梗霉的聚苹果酸产量、糖酸转化率和生产强度。【方法】采用单因素试验优化适宜出芽短梗霉BK-10菌株产生聚苹果酸的培养条件,通过Plackett-Burman法对培养基组分筛选显著性影响因素,并对其培养基中无机盐进行正交试验优化,最后进行5 L发酵罐验证。【结果】最优培养基配方和培养条件:100 g/L葡萄糖,1.5 g/L尿素,0.20 g/L KH_2PO_4,0.20 g/L ZnSO_4,0.05 g/L MgSO_4,0.75 g/L KCl,30 g/L CaCO_3,0.01%吐温-80,发酵温度26°C,250 mL摇瓶装液量50 mL。【结论】通过优化,聚苹果酸的糖酸转化率达到0.71 g/g,生产强度达到0.89 g/(L·h),较优化前分别提高了18.33%和71.15%,为发酵葡萄糖合成聚苹果酸进而生产L-苹果酸工艺的工业化生产奠定经济性基础。     

Short Communication: Effect of manganese on polysaccharide production and cellular pigmentation in the fungus Aureobasidium pullulans

Manganese supplementation resulted in higher polysaccharide levels and reduced cellular pigmentation by more than 8- or 17-fold after growth for 7d of the fungus Aureobasidium pullulans ATCC 42023 on sucrose or corn syrup, respectively, as a carbon source. The melanin content of the polysaccharide elaborated by ATCC 42023 cells also decreased if MnCl2 was added to the medium. The pullulan content of the polysaccharide synthesized by ATCC 42023 on sucrose was found to increase with increasing levels of manganese, whereas it was lower during growth on corn syrup if manganese was present.     

Polysaccharide production by a reduced pigmentation mutant of the fungus Aureobasidium pullulans

Abstract A reduced pigmentation mutant was isolated from Aureobasidium pullulans ATCC 42023 by chemical mutagenesis and was subsequently characterized. The pigment melanin was present not only in A. pullulans cells but also contaminated the elaborated polysaccharide and thus, was measured in both fractions. Cellular and polysaccharide melanin levels of the mutant strain were at least 11-fold and 18-fold reduced, respectivelu, compared toits parent strain after 7 days of growth at 30°C whether sucrose or glucose served as the carbon source in the culture medium. Polysaccharide and cell dry weight levels of the mutant were very similar to those observed for the parent after growth on sucrose or glucose as the source of carbon over a period of 7 days at 30°C. The pullulan content of the polysaccharide produced by the parent or mutant strain was lower for sucrose-grown cells than for glucose-grown cells. It was also noted that the pullulan content of the polysaccharide elaborated by the mutant strain was slightly higher than that of the polysaccharide produced by the parent strain after growth on either sucrose or glucose.     

Monitoring and control of pullulan production using vision sensor

The production of the polysaccharide pullulan by the yeast-like fungi, Aureobasidium pullulans, is accompained by cellular morphogenetic changes. High productivity and yield of the process have been found to correlate with high concentration of yeast-like cells in the culture. The morphogenetic changes of A. pullulans cells depend on the culture conditions, e.g., dissolved oxygen, shear rate and medium composition. In order to improve the productivity of the process, a novel control law was formulated. A feeding strategy dependent on the culture cellular composition was designed and aimed to keep the yeast-like cell concentration high. The culture morphogenetic composition during the process was monitored by a recently developed vision sensor. Feeding was actuated when the yeast-like cell concentration decreased below a threshold. The proposed control strategy improved pullulan production by increasing both productivity and yield of the cells by 67% and 80%, correspondingly. The results point to the advantage and the potential of using the monitoring and control system and algorithm to increase productivity and yield in cellular bioprocesses.     

Effect of nitrogen source on pullulan production by Aureobasidium pullulans grown in a batch bioreactor

Pullulan production by Aureobasidium pullulans ATCC 201253 using selected nitrogen sources was studied in a medium using corn syrup as a carbon source. Independent of the corn syrup concentration present, the use of corn steep liquor or hydrolysed soy protein as a nitrogen source instead of ammonium sulphate did not elevate polysaccharide production by ATCC 201253 cells grown in an aerated, batch bioreactor containing 4 litres of medium. Pullulan production on corn steep liquor or hydrolysed soy protein as a nitrogen source became more comparable as the concentration of corn syrup was increased. Cell weights after 7 days of growth on any of the nitrogen sources were similar. The viscosity of the polysaccharide on day 7 was highest for cells grown on ammonium sulphate and 12.5% corn syrup. The pullulan content of the polysaccharide elaborated by ammonium sulphate-grown cells on day 7 decreased as the corn syrup level rose in the medium while the pullulan content of polysaccharide produced by cells grown on corn steep liquor or soytone generally increased.     

Effect of pH on the batch fermentation of pullulan from sucrose medium

Two strains of the yeast-like fungus Aureobasidium pullulans 2552 and 140B have been used for the fermentative production of the polysaccharide pullulan from a sucrose synthetic medium. In the batch fermentation, either in Erlenmeyers or in the fermentor, the pH of the culture medium was decreased rapidly from its initial pH value of 5.5 to the self-stabilized final value of 2.5 within 24 h. Experiments on the effect of initial pH on the fermentation revealed that at very low initial pH values, such as at pH 2, the polysaccharide production was in-significant. However, the biomass concentration obtained was very high at this very low initial pH value. This interesting phenomenon was served as the basic principle for the development of the bistaged pH fermentation process for the production of pullulan. In this process the first stage of fermentation was conducted at the very acidic pH for the best production of biomass. When the biomass concentration reached its maximum value, the second stage of fermentation was initiated by adjusting the medium pH to a higher value for promoting the synthesis of the polysaccharide. Experiments conducted in Erlenmeyers and in the fermentor confirmed this concept. The bistaged pH process enhanced the polysaccharide concentration in the medium, influenced the rheological properties of the fermentation broth, and has a potential of operation under nonsterile and nonaseptic conditions.     

Flocculant and Chemical Properties of a Polysaccharide from Pullularia pullulans

An extracellular polysaccharide, PP-floc, was synthesized from glucose by Pullularia pullulans (or Aureobasidium pullulans) in a pilot plant batch fermentor containing 175 liters of culture medium. At 58 h of fermentation, the concentration of PP-floc was 1.03 g/100 ml, giving a 25.8% conversion of initial glucose to polysaccharide. The flocculant activity of the culture medium increased during the fermentation process and reached its maximum at 50 h of culture age. Less PP-floc (0.33 lb/ton of slimes [approximately 149.7 g/0.907 t]) was required to give the same flocculant activity as a synthetic polymer of acrylamide, Separan NP-10 (0.5 lb/ton of slimes [approximately 226.8 g/0.907 t]), at all temperatures from 25 to 100 C. The degree of inactivation of PP-floc and Separan NP-10 at elevated temperatures was almost identical, and they were completely inactivated at about the same temperature (80 C). PP-floc also gave better compaction of slimes than Separan NP-10 at all temperatures tested. PP-floc was soluble in water and its specific optical rotation was [alpha](D) (25) + 194 degrees in water (c, 0.4). PP-floc contained 83.3% carbohydrate, 3.2% protein, and 8.1% water. Glucose was found to be the principal sugar monomer with traces (>5%) of galactose and mannose present. Structural studies on the fractions of purified polysaccharide by methylation and by periodate oxidation techniques prove that PP-floc is linear and composed of alpha-(1 --> 4) and alpha-(1 --> 6) glucopyranosyl units in the approximate ratio of 2:1. The action of pullulanase on crude PP-floc suggested the ordered arrangement of two consecutive alpha-(1 --> 4)-linked glucopyranosyl units flanked by alpha-(1 --> 6)-linked glucopyranose residues.     

农杆菌介导的出芽短梗霉遗传转化及高效筛选聚苹果酸高产菌株

建立根癌农杆菌介导的出芽短梗霉遗传转化方法及T-DNA突变库,高效筛选聚苹果酸高产菌株及功能基因。通过含潮霉素和草铵磷抗性基因的农杆菌转化出芽短梗霉,抗性压力筛选及PCR验证建立根癌农杆菌介导的出芽短梗霉遗传转化方法,结合发酵液p H与聚苹果酸含量响应变化,微孔板高效筛选高产聚苹果酸的T-DNA插入突变株,基因组步移确定T-DNA插入位点及功能基因。结果获得遗传稳定的抗性基因菌株,每107个细胞可获得80-120个转化子,出芽短梗霉H27号T-DNA突变株聚苹果酸摇瓶发酵产量提高24.5%,基因组步移证实糖酵解途径磷酸甘油酸变位酶基因被破坏。成功建立了根癌农杆菌介导的出芽短梗霉遗传转化方法和T-DNA插入突变库,结合高效筛选方法为聚苹果酸合成功能基因挖掘及高产机制解析奠定基础。     

Polysaccharide production by a reduced pigmentation mutant of Aureobasidium pullulans NYS-1

AIMS: To isolate a reduced pigmentation mutant of Aureobasidium pullulans NYS-1 and characterize its cellular pigmentation plus its polysaccharide and biomass production relative to carbon source. METHODS AND RESULTS: Cellular pigmentation, polysaccharide levels and biomass production by the isolated mutant NYSRP-1 were analysed relative to carbon source. Cellular pigmentation of the mutant was lower than its parent strain using either carbon source. The mutant elaborated higher polysaccharide levels on sucrose than on corn syrup. The pullulan content of the polysaccharide synthesized and biomass production by the mutant rose as the carbon source concentration was increased. CONCLUSION: It is feasible to isolate a reduced pigmentation mutant from strain NYS-1 that exhibits elevated polysaccharide production using corn syrup as a carbon source. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant provides an advantage for commercial pullulan production because of its reduced pigmentation and enhanced polysaccharide synthesis.     

Modelling of simultaneous production of polygalacturonase and exopolysaccharide by Aureobasidium pullulans ATHUM 2915

The polymorphic fungus Aureobasidium pullulans ATHUM 2915, produced significant quantities of extracellular polygalacturonase and polysaccharide when grown, under controlled conditions, in liquid medium with pectin and glucose as carbon sources and nitrogen source as limited factor. Growth, substrate consumption and products formation were simulated by a structured mathematical model, which was compared with the experimental data from batch culture in a chemostat. This model was applied successfully in the study of some essential parameters influenced the process at various pH values.     

Pullulan production by a non-pigmented strain of Aureobasidium pullulans using batch and fed-batch culture

The production of pigment-free pullulan by Aureobasidium pullulans in batch and fed-batch culture was investigated. Batch culture proved to be a better fermentation system for the production of pullulan than the fed-batch culture system. A maximum polysaccharide concentration (31.3 g l⁻¹), polysaccharide productivity (4.5 g l⁻¹ per day), and sugar utilization (100%) were obtained in batch culture. In fed-batch culture, feed medium composition influenced the kinetics of fermentation. For fed-batch culture, the highest values of pullulan concentration (24.5 g l⁻¹) and pullulan productivity (3.5 g l⁻¹ per day) were obtained in culture grown with feeding substrate containing 50 g l⁻¹ sucrose and all nutrients. The molecular size of pullulan showed a decline as fermentation progressed for both fermentation systems. At the end of fermentation, the polysaccharide isolated from the fed-batch culture had a slightly higher molecular weight than that of batch culture. Structural characterization of pullulan samples (methylation and enzymic hydrolysis with pullulanase) revealed the presence of mainly α-(1→4) (∼66%) and α-(1→6) (∼31%) glucosidic linkages; however, a small amount (<3%) of triply linked (1,3,4-, 1,3,6-, 1,2,4- and 1,4,6-Glc p) residues were detected. The molecular homogeneity of the alcohol-precipitated polysaccharides from the fermentation broths as well as the structural features of pullulan were confirmed by ¹³C-NMR and pullulanase treatments followed by gel filtration chromatography of the debranched digests.     

Polysaccharide hydrolases ofAureobasidium pullulans

The polysaccharide hydrolase activity of a group of selected strains of the genus Aureobasidium pullulans was investigated using a new gel testing assay. A total of 31 strains were tested for alpha-amylase, alpha-glucosidase and glucoamylase, beta-glucosidase, lichenase, cellulase, xylanase and xylosidase, mannanase and mannosidase production during growth of microorganisms on respective meshed polysaccharide gels. Attempts were made to increase the polysaccharide hydrolase activity through selection of some A. pullulans strains by passaging them on the respective modified xylanase- and cellulase-containing gels. The individual saccharide degradation cleavage products were investigated by chromatography.     

Simultaneous production of pullulan and biosorption of metals by Aureobasidium pullulans strain CH-1 on peat hydrolysate

It was demonstrated that during the growth of Aureobasidium pullulans strain CH-1 on the acid hydrolysate of peat from the Vlasina Lake, the content of metals (Cu, Fe, Zn, Mn, Pb, Cd, Ni and Cr) decreased due to biosorption. The reduction in the metal content was found to be in the range (%): 38.2-62.2, 67.7-97.3, 0.02-62.05, 0.05-23.97, 0.16-4.24, 3.45-51.72, 1.18-35.82, 0.86-44.44, for Cu, Fe, Zn, Mn, Pb, Cd, Ni and Cr, respectively. During this process, the metals were accumulated in the biomass, while pullulan, an extracellular polysaccharide produced by Aureobasidium pullulans strain CH-1, was found not to bind the above-mentioned metals.     

Polysaccharide production by immobilized Aureobasidium pullulans cells in batch bioreactors.

Cells of the fungus Aureobasidium pullulans ATCC 201253 were entrapped within 4% agar cubes or 5% calcium alginate beads and were examined for their production of the polysaccharide pullulan in batch bioreactors. The batch bioreactors were utilized twice for 168 hours of polysaccharide production in medium containing corn syrup as a carbon source. The agar-entrapped cells produced nearly equivalent pullulan concentrations during both production cycles. The alginate-entrapped cells produced higher polysaccharide levels during the second cycle compared to the levels observed during the initial cycle. The agar-entrapped cells elaborated a polysaccharide with a higher pullulan content than did the alginate-entrapped cells during both production cycles.